CN103497917B - One strain can utilize the thermophilic Bacillus licheniformis of lignocellulosic material fermentative production 2,3-butanediol - Google Patents
One strain can utilize the thermophilic Bacillus licheniformis of lignocellulosic material fermentative production 2,3-butanediol Download PDFInfo
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Abstract
The invention discloses a strain and can utilize lignocellulosic material fermentative production 2,3-butyleneglycol is thermophilic Bacillus licheniformis, does is this bacterial strain called Bacillus licheniformis (Bacillus? licheniformis) 3F-3, it is preserved in China typical culture collection center on June 27th, 2012, does is preserving number: CCTCC? NO:M2012253.Bacterial strain of the present invention can with lignocellulosic material (wood sugar, xylose mother liquid and stalk hydrolyzed solution) for substrate, 2 are produced with the transformation efficiency higher than theoretical yield 90% under 42-55 DEG C of condition, 3-butyleneglycol, they are 2 years old, 3-butyleneglycol concentration reaches as high as 92 grams per liters, volume production is most effective reach 1.1 grams per liters/hour, there is good economic benefit and social benefit.
Description
Technical field
The present invention relates to a bacillus licheniformis, particularly relate to the thermophilic Bacillus licheniformis of fermentative production 2,3-butanediol that a strain can utilize lignocellulosic material, belong to biological technical field.
Background technology
2,3-butanediol is widely used in multiple fields (ApplMicrobiolBiotechnol, 2001,55 (1): 10-18) such as chemical industry, food, fuel and space flight and aviation.Because of its calorific value higher (27,200kJ/kg), same to ethanol (29,100kJ/kg) is suitable, can be used as fuel dope; BDO can be replaced, for the synthesis of polyester and Polyurethanes; Its dewatered product methylethylketone is a kind of low boiling point solvent, is widely used in the industries such as coating, lubricant, fuel, can be used as again the intermediate of organic synthesis spices, antioxidant etc. simultaneously; Its dewatered product 1,3-butadiene can be used to synthetic rubber monomer, is a kind of important basic petrochemical organic raw material; Form octane with methylethylketone condensation hydrogenation, can be used for producing senior aviation oil.
Current 2, the production method mainly microbe fermentation method of 3-butyleneglycol, the bacterial strain of usual employing comprises the (JBiotechnol such as Klebsiella (Klebsiella), bacillus (Bacillus) and enterobacter (Enterobacter), 1993,27 (3): 317-329).Raw material for 2,3-butanediol fermentation is mainly the raw materials such as glucose, sucrose, starch, Tapioca Starch, wherein the refined sugar such as dextrose plus saccharose, and its output is higher, all more than 150 grams per liters; But refined sugar cost is higher, be unfavorable for the suitability for industrialized production of 2,3-butanediol.
Lignocellulose is biomass renewable resources the abundantest in the world, and it is mainly made up of Mierocrystalline cellulose, hemicellulose and xylogen.At present the bacterial strain of fermenting lignocellulose raw material production 2,3-butanediol that utilizes known mostly both at home and abroad is klebsiella, but it is potential pathogenic, limits its application in 2,3-butanediol is produced.Genus bacillus has biological safety, but genus bacillus is more weak to the Utilization ability of wood sugar in lignocellulosic material, as bacillus polymyxa, it take wood sugar as fermenting substrate 24 hours 2,3-butyleneglycol output is 7.2 grams per liters (ApplMicrobiolBiotechnol, 2004,64:112 – 119); The lichen bacillus ferments wood sugar 24 hourly output is 13.8 grams per liters (BiotechnolBioeng, 2012,109:1610-1621), cannot realize industrial scale and produce.
Bacillus acidocldarius has higher leavening temperature (42 ~ 60 DEG C) due to it, greatly can reduce the probability of pollution microbes in fermenting process; , ferment with higher temperature meanwhile, can also fermentation broth viscosity be reduced, be conducive to operation or process, energy efficient and the production cost of subsequent step.But through retrieval, the thermophilic Bacillus licheniformis of lignocellulosic material fermentative production 2,3-butanediol can be utilized to have not been reported.
Summary of the invention
The deficiency of bacterial strain and fermentation process in producing for existing 2,3-butanediol, the problem to be solved in the present invention is to provide the thermophilic Bacillus licheniformis of fermentative production 2,3-butanediol that a strain can utilize lignocellulosic material.
The fermentative production 2 utilizing lignocellulosic material of the present invention, 3-butyleneglycol is thermophilic Bacillus licheniformis, it is characterized in that: this bacterial strain is called Bacillus licheniformis (Bacilluslicheniformis) 3F-3, it is preserved in China typical culture collection center on June 27th, 2012 and (is called for short CCTCC, Luojiashan, Wuchang, Wuhan City, Hubei Province), preserving number is: CCTCCNO:M2012253.
The thermophilic Bacillus licheniformis of fermentative production 2,3-butanediol of above-mentioned utilized lignocellulosic material, its biological property is: form is bar shape, long 1.5 μm ~ 3.0 μm, diameter 0.6 μm ~ 0.7 μm, and colony colour is yellow or white, produces gemma; Physiological and biochemical property is: VP reaction is positive, glucose, sucrose, fructose, wood sugar can be utilized to produce acid, and hydrolyzable casein, gelatin, tween 80, can utilize Citrate trianion, can grow in containing the substratum of 70 grams per liter NaCl, can grow under 42 ~ 60 DEG C of conditions; 16SrDNA sequence length is 1369bp, and its nucleotide sequence is as shown in SEQIDNO.1.
The 16SrDNA sequence of the thermophilic Bacillus licheniformis of fermentative production 2,3-butanediol of above-mentioned utilized lignocellulosic material and the 16SrDNA sequence of other known Bacillus licheniformis have the similarity of 99 ~ 100%.
Thermophilic Bacillus licheniformis of the present invention is utilizing the application in lignocellulosic material fermentative production 2,3-butanediol.
Thermophilic Bacillus licheniformis CCTCCNO:M2012253 of the present invention is first accessed in seed culture medium in conventional manner, cultivate 8 ~ 12 hours at 42 ~ 55 DEG C, and then transfer in conventional manner into fermention medium, fermentation condition is: culture temperature 42 ~ 55 DEG C, and training method is stir culture (air flow is 0.5vvm ~ 1.0vvm), mixing speed 200 ~ 500 revs/min, rotation radius is 33mm, incubation time 18 ~ 60 hours, the obtained fermented liquid containing 2,3-butanediol.
By fermented liquid 8,000 rev/min centrifugal 10 minutes, gets supernatant liquor dilution, detect 2,3-butanediol content.
Contain during above-mentioned seed culture medium often rises: wood sugar 30 ~ 50 grams, yeast powder 5 ~ 10 grams, Dried Corn Steep Liquor Powder 5 ~ 15 grams, potassium primary phosphate 4 grams, sodium acetate 2 ~ 6 grams, 1 ~ 2 gram, Repone K, 0.2 gram, magnesium sulfate, surplus is water; PH is 6 ~ 7.115 DEG C of sterilizings 20 minutes.
Contain during above-mentioned fermention medium often rises: yeast powder 5 ~ 20 grams, peptone 5 ~ 20 grams, Dried Corn Steep Liquor Powder 5 ~ 20 grams, soybean peptides 5 ~ 20 grams, at least one in soy peptone 5 ~ 20 grams of five kinds of nitrogenous sources; Also contain in often liter of fermention medium in addition: lignocellulosic material (as wood sugar, stalk hydrolyzed solution or xylose mother liquid etc.) 80 ~ 160mL, 500 grams per liters are no less than containing first sugar amount in its Raw, potassium primary phosphate 4 grams, sodium acetate 1 ~ 6 gram, 2 grams, Repone K, 0.2 gram, magnesium sulfate, all the other are water; PH is 6 ~ 7.115 DEG C of sterilizings 20 minutes.
The preferred culture temperature of above-mentioned fermentation condition 50 ~ 55 DEG C, incubation time 18 ~ 48 hours.
Feature of the present invention and beneficial effect are: thermophilic Bacillus licheniformis CCTCCNO:M2012253 provided by the invention is Biosafety bacterial strain, can utilize the glucose and xylose in lignocellulosic material simultaneously; This bacterial strain take lignocellulosic material as substrate, and the 2,3-butanediol maximum concentration obtained at 42 ~ 55 DEG C of condition bottom fermentations can reach 92 grams per liters, production intensity can reach 1.1 grams per liters/hour; Compare the existing obvious output of 2,3-butanediol bacterial strain of producing high, production intensity is large; Thermophilic Bacillus licheniformis CCTCCNO:M2012253 provided by the invention is in production 2, in 3-butyleneglycol, utilize the advantage of thermophilic fermentation process, the probability of pollution microbes is little, and make full use of lignocellulosic material, meet the state basic policy of Sustainable development, there is good economic benefit and social benefit.
Embodiment
Illustrate the present invention further below by embodiment, but be not limited only to this.
Embodiment 1: utilize wood-sugar fermentation to produce 2,3-butanediol and screen thermophilic lichem bacillus strain
Obtain soil sample from orchard and other places, soak 2 hours by screening culture medium, in 500mL Erlenmeyer flask, fill soak solution 100mL, under 50 DEG C of conditions, be placed on shaking table and cultivate 24 hours with the rotating speed of 180 revs/min.By nutrient solution with 10
-2, 10
-3, 10
-4, 10
-5, 10
-6five extent of dilution are coated in the culture dish of solid screening culture medium, cultivate 12 hours for 50 DEG C.Choosing single bacterium colony, enlarged culturing, take wood sugar as carbon source, carries out primary dcreening operation by VP reaction.Positive strain is filtered out according to reaction times and colour-change.Screening principle be that 2,3-butanediol is oxidized to di-acetyl in the basic conditions, with red material synthesized by creatine or guanidine derivatives, add naphthyl alcohol, creatine can promote reaction.Then measure the 2,3-butanediol output of the positive bacteria of primary dcreening operation, select the bacterial strain of wherein production peak.
The bacterial strain that output obtained above is the highest is Bacillus licheniformis 3F-3.
Above-mentioned screening and culturing based formulas is: contain in 1L distilled water: 50 grams of wood sugars, 10 grams of peptones, 5 grams of yeast powders, 10 grams of NaCl, sterilizing 20 minutes under 115 DEG C of conditions.Solid screening culture medium is the agar powder adding 20 grams per liters on the basis of above-mentioned formula.
Above-mentioned Bacillus licheniformis 3F-3 strain morphology is bar shape, long 1.5 μm ~ 3.0 μm, diameter 0.6 μm ~ 0.7 μm, colony colour is yellow or white, produces gemma, VP reaction is positive, glucose, sucrose, fructose, wood sugar can be utilized to produce acid, and hydrolyzable casein, gelatin, tween 80, can utilize Citrate trianion, can grow in containing the substratum of 70 grams per liter NaCl, can grow under 42 ~ 60 DEG C of conditions.Its 16SrDNA sequence length is 1369bp, and nucleotide sequence, as shown in SEQIDNO.1, has the similarity of 99 ~ 100% with the 16SrDNA sequence of other known Bacillus licheniformis.
Above-mentioned bacterial strains is called Bacillus licheniformis (Bacilluslicheniformis) 3F-3, and it is preserved in China typical culture collection center on June 27th, 2012, and preserving number is: CCTCCNO:M2012253.
Embodiment 2: the cellular liquid culture of preparation Bacillus licheniformis CCTCCNO:M2012253 bacterial strain
Bacillus licheniformis CCTCCNO:M2012253 bacterial strain one ring that the sugary LB medium slant of picking solid is cultivated, be seeded in the 500mL Erlenmeyer flask of the 100mL seed culture medium that sterilizing is housed, under 50 DEG C of conditions, be placed on shaking table and cultivate 12 hours with the rotating speed of 180 revs/min, be i.e. the cellular liquid culture of obtained Bacillus licheniformis CCTCCNO:M2012253 bacterial strain.
Above-mentioned sugary solid LB media often rises and contains: 40 grams of wood sugars, 10 grams of peptones, 5 grams of yeast powders, 10 grams of NaCl, 20 grams of agar powders, sterilizing 20 minutes under 115 DEG C of conditions.
Above-mentioned seed culture medium often rises and contains: wood sugar 50 grams, yeast powder 5 grams, Dried Corn Steep Liquor Powder 15 grams, potassium primary phosphate 4 grams, sodium acetate 2 grams, 1 gram, Repone K, 0.2 gram, magnesium sulfate, and surplus is water; The pH of described seed culture medium is sterilizing 20 minutes under 6 ~ 7,115 DEG C of conditions.
Embodiment 3: utilize Bacillus licheniformis CCTCCNO:M2012253 bacterial strain to produce 2,3-butanediol with fermention medium 1 in 5L fermentation cylinder for fermentation
By the cellular liquid culture of the Bacillus licheniformis CCTCCNO:M2012253 bacterial strain that the method by embodiment 2 obtains, be seeded in the inoculum size of volume ratio 10% in the 5L fermentor tank of the 4L fermention medium that sterilizing is housed, under 50 DEG C of conditions, fermentor tank mixing speed 400 revs/min, air flow is 0.5vvm ~ 1.0vvm, DNS method detects Xylose Content, when xylose concentration adds wood sugar lower than during 20 grams per liter, ferment 36 hours, the 2,3-butanediol fermented liquid that concentration is 75 ~ 92 grams per liters can be obtained.
Above-mentioned fermention medium 1 is filled a prescription as follows:
Wood sugar 60 grams per liter, yeast powder 10 grams per liter, Dried Corn Steep Liquor Powder 15 grams per liter, potassium primary phosphate 4 grams per liter, sodium acetate 2 grams per liter, Repone K 2 grams per liter, 0.2 gram, magnesium sulfate.Sterilizing 20 minutes under 115 DEG C of conditions.
Embodiment 4: utilize Bacillus licheniformis CCTCCNO:M2012253 bacterial strain to produce 2,3-butanediol with fermention medium 2 in 5L fermentation cylinder for fermentation
By the cellular liquid culture of the Bacillus licheniformis CCTCCNO:M2012253 bacterial strain that the method by embodiment 2 obtains, be seeded in the inoculum size of volume ratio 10% in the 5L fermentor tank of the 4L fermention medium that sterilizing is housed, under 50 DEG C of conditions, fermentor tank mixing speed 400 revs/min, air flow is 0.5vvm ~ 1.0vvm, DNS method detects total sugar content (glucose and xylose), total sugar concentration adds corn stalk hydrolysis lower than during 20 grams per liter, ferment 48 hours, the 2,3-butanediol fermented liquid that concentration is 65 ~ 73 grams per liters can be obtained.
Above-mentioned fermention medium 2 is filled a prescription as follows:
Corn stalk hydrolysis 100ml/ liter, yeast powder 10 grams per liter, Dried Corn Steep Liquor Powder 10 grams per liter, potassium primary phosphate 4 grams per liter, sodium acetate 1 grams per liter, Repone K 2 grams per liter, 0.2 gram, magnesium sulfate.Sterilizing 20 minutes under 115 DEG C of conditions.
Wherein corn stalk hydrolysis contains glucose 400 grams per liter, and wood sugar 115 grams per liter, for Changchun, great achievement new resources Group Co., Ltd provides.
Embodiment 5: utilize Bacillus licheniformis CCTCCNO:M2012253 bacterial strain to produce 2,3-butanediol with fermention medium 3 in 5L fermentation cylinder for fermentation
Repeat the operation of embodiment 3, just change fermention medium 1 into fermention medium 3, the concentration of fermenting to the 36th hour 2,3-butanediol reaches 61.0g/L.
Above-mentioned fermention medium 3 is filled a prescription as follows:
Corn stalk hydrolysis 120ml/ liter, Dried Corn Steep Liquor Powder 15 grams per liter, potassium primary phosphate 4 grams per liter, sodium acetate 6 grams per liter, Repone K 2 grams per liter, 0.2 gram, magnesium sulfate.Sterilizing 20 minutes under 115 DEG C of conditions.
Wherein corn stalk hydrolysis provides for Changchun great achievement new resources Group Co., Ltd.
Embodiment 6: utilize Bacillus licheniformis CCTCCNO:M2012253 bacterial strain to produce 2,3-butanediol with fermention medium 4 in 5L fermentation cylinder for fermentation
Repeat the operation of embodiment 3, just change fermention medium 1 into fermention medium 4, the concentration of fermenting to the 54th hour 2,3-butanediol reaches 57.0g/L.
Above-mentioned fermention medium 4 is filled a prescription as follows:
Xylose mother liquid 80ml/ liter, Dried Corn Steep Liquor Powder 15 grams per liter, peptone 5 grams per liter, potassium primary phosphate 4 grams per liter, sodium acetate 3 grams per liter, Repone K 2 grams per liter, 0.2 gram, magnesium sulfate.Sterilizing 20 minutes under 115 DEG C of conditions.
Wherein, containing glucose 90 grams per liter in xylose mother liquid, wood sugar 450 grams per liter, pectinose 146 grams per liter; Shandong Longli Biology Science and Technology Co., Ltd provides.
Claims (1)
1. a strain can utilize lignocellulosic material fermentative production 2, the thermophilic Bacillus licheniformis of 3-butyleneglycol, it is characterized in that: this bacterial strain is called Bacillus licheniformis (Bacilluslicheniformis) 3F-3, it is preserved in China typical culture collection center on June 27th, 2012, and preserving number is: CCTCCNO:M2012253.
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| EP3543344B1 (en) | 2018-03-22 | 2023-06-28 | Biopolis, S.L. | Procedure for obtaining 2,3-butanediol by fermentation of hydrolyzed substrates of organic waste (ow) with microorganisms |
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| CN101851598A (en) * | 2010-05-10 | 2010-10-06 | 江南大学 | Environmentally-friendly breeding of bacillus subtilis for producing 2,3-butanediol by fermentation with glucose substrate |
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